Amy K. Cain

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Approaches to querying bacterial genomes with transposon-insertion sequencing

In this review, we discuss transposon-insertion sequencing, variously known in the literature as TraDIS, Tn-seq, INSeq, and HITS. By monitoring a large library of single transposon-insertion mutants with high-throughput sequencing, these methods can rapidly identify genomic regions that contribute to organismal fitness under any condition assayable in the laboratory with exquisite resolution. We discuss the various protocols that have been developed and methods for analysis. We provide an overview of studies that have examined the reproducibility and accuracy of these methods, as well as studies showing the advantages offered by the high resolution and dynamic range of high-throughput sequencing over previous methods. We review a number of applications in the literature, from predicting genes essential for in vitro growth to directly assaying requirements for survival under infective conditions in vivo. We also highlight recent progress in assaying non-coding regions of the genome in addition to known coding sequences, including the combining of RNA-seq with high-throughput transposon mutagenesis.

Transposons related to Tn1696 in IncHI2 plasmids in multiply antibiotic resistant Salmonella enterica serovar Typhimurium from Australian animals

Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, bla(TEM), and aphA1 genes conferring resistance to streptomycin, sulphonamides, ampicillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 and Tn6029 overlap, sharing one IS26 copy. This suggests that they do not move by a standard transpositional mechanism. A circular intermediate, carrying only the region containing the resistance gene(s) and one of the IS26 bounding it, is proposed as an intermediate.

High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile

ABSTRACT Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection. Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.

A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae.

Multidrug‐resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource‐poor hospital settings. Through whole‐genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high‐dependency wards in a hospital in Kathmandu during 2012 with a case‐fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage‐specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward‐specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM‐1 containing lineage in 2011 would have alerted the high‐dependency ward staff to intervene. We argue that some form of real‐time genetic characterisation, alongside clade‐specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high‐ and low‐income settings.

Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ

OBJECTIVES To determine the structure of the resistance region in an IncHI1 plasmid conferring resistance to multiple antibiotics, including gentamicin, recovered from a Salmonella enterica serovar Typhimurium isolate from a horse. METHODS Plasmids were recovered by conjugation. The plasmid type, resistance genes and their context were identified by PCR, cloning, hybridization and DNA sequencing. The sequence was compared using bioinformatic tools with available resistance region sequences. RESULTS In isolate SRC27, an IncI1 plasmid, pSRC27-I, conferred streptomycin resistance via the strA and strB genes contained within Tn5393a. An IncHI1 plasmid, pSRC27-H, was found to carry the aacC2 gentamicin resistance gene within a 34.6 kb multiple antibiotic resistance region that included nine further antibiotic resistance genes, aadA2, aphA1, bla(TEM), catA1, dfrA12, strA and strB, sul2 and tetA(B), conferring resistance to streptomycin and spectinomycin, kanamycin and neomycin, ampicillin, chloramphenicol, trimethoprim, streptomycin, sulfamethoxazole and tetracycline, respectively. This complex resistance region has evolved from Tn2670 and Tn10 via loss and gain of DNA segments. It includes Tn6029 and Tn4352, and a new transposon carrying the aacC2 gene. It also contains five copies of IS26, two of IS1 and one each of IS10 and ISCfr1. This region of pSRC27-H is related to ones present at the same position in three sequenced IncHI1 plasmids, pHCM1, pO111_1 and pMAK1, but has acquired new segments carrying antibiotic resistance genes. CONCLUSIONS Evolution via loss and gain of resistance genes has occurred within the large resistance region of pHCM1-type IncHI1 plasmids leading to different resistance phenotypes.

An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid

The population of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, exhibits limited DNA sequence variation, which complicates efforts to rationally discriminate individual isolates. Here we utilize data from whole-genome sequences (WGS) of nearly 2,000 isolates sourced from over 60 countries to generate a robust genotyping scheme that is phylogenetically informative and compatible with a range of assays. These data show that, with the exception of the rapidly disseminating H58 subclade (now designated genotype 4.3.1), the global S. Typhi population is highly structured and includes dozens of subclades that display geographical restriction. The genotyping approach presented here can be used to interrogate local S. Typhi populations and help identify recent introductions of S. Typhi into new or previously endemic locations, providing information on their likely geographical source. This approach can be used to classify clinical isolates and provides a universal framework for further experimental investigations.

The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries

Summary: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. Availability and implementation: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-Tradis Contact: parkhill@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Evolution of IncHI2 plasmids via acquisition of transposons carrying antibiotic resistance determinants.

OBJECTIVES To investigate the relationships between IncHI2 plasmids conferring resistance to antibiotics isolated in Australia and those from other countries. METHODS PCR, restriction digestion, cloning and DNA sequencing were used to characterize transposons and determine their location in IncHI2 plasmids recovered from Salmonella enterica isolates from Australian animals. RESULTS Tn10, carrying the tet(B) tetracycline resistance determinant, was found in IncHI2 plasmids pSRC26 and pSRC125 recovered from S. enterica serovar Typhimurium from Australian cattle and in IncHI2 plasmids from serovar Infantis isolates from chickens. Its location was the same as seen in the IncHI2 reference plasmid R478. The location of Tn1696-related mercury and multiple antibiotic resistance transposons was also the same in all of the Australian plasmids, and the mer end was in the same position as the mer module in R478. However, R478 has lost the tnp end (including most of the integron) and some adjacent sequence. The sequence adjacent to the tnp end of the Tn1696-related transposons in the Australian plasmids is in the bla(CMY-8)-carrying plasmid pK29, but only 22 bp from the transposon inverted repeat remains. These plasmids all belong to the same evolutionary lineage. Neither transposon was found in TP116, which represents a separate lineage. CONCLUSIONS Transposon locations are useful markers for lineages of closely related plasmids. The configuration surrounding the Tn1696-like transposons in the Australian IncHI2 plasmids is ancestral to those found in R478 and pK29, each of which has part of the transposon and adjacent sequence replaced by other resistance genes.

Emergence and Evolution of Multiply Antibiotic-Resistant Salmonella enterica Serovar Paratyphi B d-Tartrate-Utilizing Strains Containing SGI1

ABSTRACT The first Australian isolate of Salmonella enterica serovar Paratyphi B d-tartrate-utilizing (dT+) that is resistant to ampicillin, chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamides, and tetracycline (ApCmFlSmSpSuTc) and contains SGI1 was isolated from a patient with gastroenteritis in early 1995. This is the earliest reported isolation globally. The incidence of infections caused by these SGI1-containing multiply antibiotic-resistant S. enterica serovar Paratyphi B dT+ strains increased during the next few years and occurred sporadically in all states of Australia. Several molecular criteria were used to show that the early isolates are very closely related to one another and to strains isolated during the following few years and in 2000 and 2003 from home aquariums and their owners. Early isolates from travelers returning from Indonesia shared the same features. Thus, they appear to represent a true clone arising from a single cell that acquired SGI1. Some minor differences in the resistance profiles and molecular profiles also were observed, indicating the ongoing evolution of the clone, and phage type differences were common, indicating that this is not a useful epidemiological marker over time. Three isolates from 1995, 1998, and 1999 contained a complete sul1 gene but were susceptible to sulfamethoxazole due to a point mutation that creates a premature termination codon. This SGI1 type was designated SGI1-R. The loss of resistance genes also was examined. When strains were grown for many generations in the absence of antibiotic selection, the loss of SGI1 was not detected. However, variants SGI1-C (resistance profile SmSpSu) and SGI1-B (resistant to ApSu), which had lost part of the integron, arose spontaneously, presumably via homologous recombination between duplications in the In104 complex integron.